A multi-part research program has been designed to yield information about the expression of the transfer DNA genes in E. coli, the biosynthesis and role of modified nucleotides in E. coli tRNA, and the organization, structure, and expression of the genes which code for the metabolically stable E. coil 4.5S and 6S RNAs. In an effort to identify tRNA species whose synthesis and rate of turnover are regulated differently from that of bulk tRNA, a two-dimensional acrylamide gel electrophoresis procedure will be used to determine the levels of individual tRNA species in cells in a variety of metabolic states. The fine structure and transcriptional properties of the two gene systems for the major E. coli leucine tRNA will be determined from DNA sequencing and in vivo and in vitro transcription experiments in which cloned tDNA will be used. Also, physiological effects of increasing the cellular dosage of tRNALeu genes will be characterized. Finally, cloned 4.5S and 6S RNA genes will be sequenced and in vivo and in vitro expression studies undertaken to characterize the gene systems; the maturation of the precursor RNAs derived from each and most important gain insight into the biological role of these RNAs.